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ABSTRACT: Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of d-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that d-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type β-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.
Journal of Biological Chemistry 07/2012; 287(37):31061-72. · 4.77 Impact Factor
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ABSTRACT: Congerin is a proto-type galectin distributed on the skin and mucosal epithelia of the upper digestive tract of the Japanese conger eel Conger myriaster. It has at least 2 isotypes, namely, congerin I and II, and plays a role in bio-defense at the body surface. In the current study, we identified both isotypes in the peritoneal fluid and peritoneal cells of C. myriaster by western blot and mass spectrometry (MS)/MS analysis. Cucullanus nematodes parasitize the abdominal cavity of C. myriaster, and immunohistochemical analyses demonstrated that congerins can bind to both the body surface of the encapsulated nematodes and the encapsulating cells. Furthermore, adhesion of the peritoneal cells to Sepharose particles was greatly accelerated when the microspheres were coated with congerin. Indeed, this effect was significantly hampered by the addition of lactose. These results indicate that congerin participates in the cellular encapsulation of the Cucullanus nematode via the induction of cellular adhesion to the parasites depending on lectin-glycoside recognition.
Fish & Shellfish Immunology 07/2012; 33(4):780-7. · 3.32 Impact Factor
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ABSTRACT: Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin
under their optimal conditions for 24h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide
mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method.
Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates
showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and
prolamin. The globulin hydrolysate (Opep2) from 2h-pepsin hydrolysis was separated by using three consecutive purification
steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase
protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6–30 amino acid residues with molecular
masses ranging from 670–3,611Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.
European Food Research and Technology 04/2012; 228(4):553-563. · 1.57 Impact Factor
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ABSTRACT: Angiotensin-I-converting enzyme (ACE) plays a crucial role in the crisis of hypertension. Some peptides that originate from
protease hydrolysates are known to suppress ACE activity in vitro and in vivo. Here, we investigated whether trypsin hydrolysate
of oyster Crassostrea gigas showed hypotensive activity and ACE inhibition. The hydrolysate significantly suppressed systolic blood pressure and ACE
activity in spontaneously hypertensive rats following a one-shot oral administration and a long-term feeding experiment lasting
9weeks. Each hydrolysate from oyster tissue showed ACE inhibitory activity, indicating the hypotensive effect was due to
synergism. One potent ACE inhibitory peptide, Asp-Leu-Thr-Asp-Tyr, was identified from the hydrolysate of the striate muscle,
and the peptide exhibited hypotensive activity in vivo. Protease digestion analysis suggested that Asp-Tyr could be the real
effector of this penta-peptide in vivo.
KeywordsAngiotensin-I-converting enzyme-Hypotensive-Oyster-Peptide-Spontaneously hypertensive rat
Fisheries Science 04/2012; 76(5):865-872. · 0.94 Impact Factor
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ABSTRACT: Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families.
Journal of amino acids. 01/2011; 2011:838914.
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ABSTRACT: Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).
Meat Science 10/2010; 87(3):196-201. · 2.28 Impact Factor
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The Journal of Immunology 04/2010; 184(7):4042. · 5.79 Impact Factor
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The Journal of Immunology 04/2010; 184(7):4043. · 5.79 Impact Factor
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ABSTRACT: Conger eel galectins, congerin I (ConI) and congerin II (ConII), show the different molecular characteristics resulting from accelerating evolution. We recently reconstructed a probable ancestral form of congerins, Con-anc. It showed properties similar to those of ConII in terms of thermostability and carbohydrate recognition specificity, although it shares a higher sequence similarity with ConI than ConII.
In this study, we have focused on the different amino acid residues between Con-anc and ConI, and have performed the protein engineering of Con-anc through site-directed mutagenesis, followed by the molecular evolution analysis of the mutants. This approach revealed the functional importance of loop structures of congerins: (1) N- and C-terminal and loop 5 regions that are involved in conferring a high thermostability to ConI; (2) loops 3, 5, and 6 that are responsible for stronger binding of ConI to most sugars; and (3) loops 5 and 6, and Thr38 residue in loop 3 contribute the specificity of ConI toward lacto-N-fucopentaose-containing sugars.
Thus, this methodology, with tracing of the molecular evolution using ancestral mutants, is a powerful tool for the analysis of not only the molecular evolutionary process, but also the structural elements of a protein responsible for its various functions.
BMC Evolutionary Biology 02/2010; 10:43. · 3.52 Impact Factor
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ABSTRACT: The crystal structure of the L-rhamnose-binding lectin CSL3 was determined to 1.8 A resolution. This protein is a component of the germline-encoded pattern recognition proteins in innate immunity. CSL3 is a homodimer of two 20 kDa subunits with a dumbbell-like shape overall, in which the N- and C-terminal domains of different subunits form lobe structures connected with flexible linker peptides. The complex structures of the protein with specific carbohydrates demonstrated the importance of the most variable loop region among homologues for the specificity toward oligosaccharides. CSL3 and Shiga-like toxin both use Gb(3) as a cellular receptor to evoke apoptosis. They have very different overall architecture but share the separation distance between carbohydrate-binding sites. An inspection of the structure database suggested that the pseudo-tetrameric structure of CSL3 was unique among the known lectins. This architecture implies this protein might provide a unique tool for further investigations into the relationships between architecture and function of pattern recognition proteins.
Journal of Molecular Biology 07/2009; 391(2):390-403. · 4.00 Impact Factor
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ABSTRACT: A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 degrees C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (K(m) and V(max) values) were calculated from Lineweaver-Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 07/2009; 154(2):229-34. · 1.61 Impact Factor
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ABSTRACT: The amino acid sequence of mannose-binding lectin, named DB1, from the yam (Dioscorea batatas, synonym Dioscorea polystachya) tubers was determined. The lectin was composed of two isoforms DB1(Cys86) and DB1(Leu86) consisting of 108 amino acid residues with 90% sequence homology between them. DB1 showed a high sequence similarity to snowdrop (Galanthus nivalis) bulb lectin, GNA; especially, the carbohydrate-binding sites of GNA were highly conserved in DB1. DB1 interacted with D-mannose residues of oligosaccharides, and the oligosaccharides carrying two mannose-alpha-1,3-D-mannose units showed high binding affinity. DB1 was examined for insecticidal activity against Helicoverpa armigera (Lepidoptera: Noctuidae) larvae at different stages of development. The rate of adults successfully emerging from pupae fed on DB1 was 33%, when incorporated into an artificial diet at a level of 0.01% (w/w). Although DB1 had no or marginal inhibitory effects on gut proteolytic and glycolic enzymes, the lectin strongly bound to larval brush border and peritrophic membrane detected by immunostaining. The results show that DB1 may fulfill a defense role against insect pests.
Journal of Agricultural and Food Chemistry 05/2009; 57(7):2896-902. · 2.82 Impact Factor
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ABSTRACT: Rice bran contains underutilised protein materials. Sequential extraction of rice bran protein (RBP) from defatted rice bran
was conducted based on the differences in their solubility. Three extraction methods were investigated. Method 1 involved
the isoelectric and acetone precipitation using water, 50gkg−1 NaCl, 0.02molL−1 NaOH and 70% ethanol as extracting solvents for albumin (pH 4.1), globulin (pH 4.3), glutelin (pH 4.8) and prolamin, respectively.
Method 2 adopted dialysis and sequential extraction was carried out with 20gkg−1 NaCl, 70% ethanol, 0.1molL−1 acetic acid and 0.1molL−1 NaOH solution as extracting solvents. Method 3 combined dialysis, isoelectric and acetone precipitation for the extraction.
Based on the yields and data obtained from sodium dodecyl sulphate polyacrylamide gel electrophoresis, size-exclusion chromatography
and differential scanning calorimetry, method 3 was chosen for the isolation and characterization of RBPs. Rice bran protein
fraction (RBPF)—albumin, globulin, glutelin and prolamin were obtained in good yields. Denaturation temperature and enthalpy
values of denaturation of RBPF vary. Highest phytate content was found in albumin and lowest in prolamin. The highest antioxidative
and hemagglutinating activities were observed in albumin.
European Food Research and Technology 12/2008; 228(3):391-401. · 1.57 Impact Factor
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Yasuharu Watanabe,
Hiroaki Tateno,
Sachiko Nakamura-Tsuruta,
Junko Kominami,
Jun Hirabayashi,
Osamu Nakamura,
Tasuku Watanabe,
Hisao Kamiya,
Takako Naganuma,
Tomohisa Ogawa,
Ryno J Naudé, Koji Muramoto
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ABSTRACT: L-rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3, on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs.
Developmental & Comparative Immunology 10/2008; 33(2):187-97. · 3.27 Impact Factor
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ABSTRACT: Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-alpha production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-alpha levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-alpha were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-alpha expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-alpha production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-kappaB family or AP-1. The TNF-alpha induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-alpha production or that biotin excess down-regulates TNF-alpha production, suggesting that biotin status may influence inflammatory diseases.
Journal of Leukocyte Biology 05/2008; 83(4):912-20. · 4.99 Impact Factor
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ABSTRACT: A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A(2) ([Lys(49)]PLA(2)), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as alpha-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for alpha-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.
Protein Expression and Purification 05/2008; 58(2):194-202. · 1.59 Impact Factor
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ABSTRACT: By using EDTA and a trypsin solution, we established a method for isolating the epidermal cells of the conger eel, Conger myriaster. We then identified TNF decoy receptor (DcR) cDNA in the species from a suppression subtractive hybridization library prepared from the epidermal cells stimulated with LPS. The full-length cDNA of conger TNF DcR (conDcR) consisted of 1479 base pairs, and the protein comprised 286 amino acid residues. Phylogenetic analysis indicated that conDcR was clustered into a DcR3 branch. ConDcR is likely to act as an important immune-regulating factor in inhibiting the apoptosis-inducing effect of TNF in the skin of conger eel.
Fish & Shellfish Immunology 04/2008; 24(3):366-71. · 3.32 Impact Factor
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ABSTRACT: The modulating effect of invertebrate C-type lectins on the crystallization of calcium carbonate was investigated. The multiple C-type lectins, named BRA-1, -2 and -3, isolated from the acorn barnacle Megabalanus rosa inhibited the nucleation and growth of calcium carbonate crystals. Among BRA, BRA-2 most efficiently inhibited the crystal nucleation of aragonite and calcite at the concentrations of > 3.3 and > 26 μg/mL, respectively. The inhibitory activities of BRA were enhanced by the addition of various biomolecules such as D-glucosamine, D-galactosamine, chitosan oligosaccharides, L-arginine and L-aspartic acid. Although the crystals of aragonite and calcite were formed in the presence of lower concentrations of BRA, the shape and size of the crystals were changed. These results further indicate the participation of the lectins in biomineralization.
Fisheries Science 03/2008; 74(2):418 - 424. · 0.94 Impact Factor
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Yasuharu Watanabe,
Nobuyuki Shiina,
Fuminori Shinozaki,
Hiroshi Yokoyama,
Junko Kominami,
Sachiko Nakamura-Tsuruta,
Jun Hirabayashi,
Kazuhiro Sugahara,
Hisao Kamiya,
Hiroki Matsubara,
Tomohisa Ogawa, Koji Muramoto
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ABSTRACT: A rhamnose-binding lectin, named SFL, was isolated from the eggs of ayu (sweet fish, Plecoglossus altivelis) by affinity and ion-exchange chromatographies. SFL revealed 287 amino acid residues with 3 tandemly repeated domains, and contained 8 half-Cys residues in each domain. The lectin was shown to have a highly specific binding affinity to globotriaosylceramide (Gb3) by frontal affinity chromatography using 100 oligosaccharides. SFL was localized in several tissues and serum of both male and female ayu, such as gill, liver, ovary, testis, intestine, stomach, brain, kidney and serum. The lectin agglutinated the spores of the microsporidian Glugea plecoglossi, which is a pathogen of ayu. Although SFL bound to glycoproteins and glycolipids of G. plecoglossi spores, Gb3 could not be detected in either of them. The results suggest that SFL could interact with various glycoconjugates of pathogens to play a role in the adhesion of microorganisms invading in the body.
Developmental & Comparative Immunology 02/2008; 32(5):487-99. · 3.27 Impact Factor
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ABSTRACT: The development of degradable and edible films from protein sources has drawn significant attention for the utilisation of natural resources as well as for the alleviation of the environmental burden. Rice bran protein (RBP) was applied to protein film preparation in this study. The protein solutions were casted on plastic tissue culture dishes with glycerol as a plasticiser after heat treatment. Functional properties of the films were then measured. The puncture strength (PS) of RBP films increased up to pH 8.0 and then decreased. PS of protein films depends on the degree of protein purity, quality and composition. Higher concentration of glycerol weakened the films. The pH affected the water solubility of RBP films and the films showed least solubility at pH 3.0. RBP could be utilised in the preparation of degradable protein-based films. The RBP-based film had functional properties comparable to those of the soy protein-based ones.
International Journal of Food Science & Technology 01/2008; 43(3):476 - 483. · 1.26 Impact Factor
